Validation of an immunohistochemistry method for detection of immature germ cells in semen from men with non-obstructive azoospermia.

Lingen, M. van (2013) Validation of an immunohistochemistry method for detection of immature germ cells in semen from men with non-obstructive azoospermia. thesis, Medicine.

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Abstract

Introduction: In 10-15% of infertile men the cause is azoospermia. There are two types of azoospermia, obstructive and non-obstructive azoospermia. Obstructive azoospermia (OA) is caused by an obstruction of the bilateral ducts. No spermatozoa or immature germ cells can be present in semen. Non-obstructive azoospermia (NOA) is caused by primary or secondary testicular failure, but immature germ cells may be present in semen. Presence of immature germ cells in semen represents little spermatogenesis of the testis. Together with somatic cells they are defined as ‘round cells’ found in semen. The exact content of these round cells is still difficult to determine. With a specific germ cell marker, like VASA, it could be possible to detect immature germ cells in semen. So far known VASA protein is specifically expressed in the germ cell lineage. Men with NOA can only have biological children through Testicular sperm extraction (TESE) and sperm retrieval rates vary between the 45 % and 63 %. Because some 50 % of men had surgery without finding spermatozoa, but could suffer complications of surgery, a non-invasive predictor of TESE outcome would be beneficial. With detecting the presence of germ cells with a specific germ cell marker we want to investigate if this can predict TESE outcome and may serve as a non-invasive predictor. Objective: The primary aim of this study is to detect immature germ cells in semen from men with azoospermia through immunohistochemistry by expressing VASA and thereby serve as a non-invasive parameter to predict the outcome of TESE. Methods: An observational retrospective cohort study was performed using semen samples collected at the outpatient clinic of Andrology of the Erasmus Medical Centre from 84 men with OA and 131 men with NOA. Inclusion took place between January 2009 and September 2012. Also 5 semen samples of men with normospermia and oligozoospermia were obtained to define the normal situation. From all semen samples the cells were fixed onto a microscope slide using a cytospin machine. Afterwards immunohistochemistry was done using the polyclonal antibody, VASA. Slides of testis tissue were obtained from 4 men with azoospermia and 1 man with normospermia and were used to define that VASA indeed is germ cell specific in testis tissue. Criteria of the different kind of germ cells were defined by a specialized test panel and afterwards all slides were assessed under a light microscope. Results: Our primary results showed that it is possible to detect immature germ cells in semen from men with NOA by expressing the VASA protein through immunohistochemistry. It was possible to distinguish spermatocytes and spermatids. In the semen from men with NOA the mean number of immature germ cells ranged from zero to a few thousand. In semen some epithelial cells also expressed VASA. Therefore immunohistochemistry was done on slides of the vesiculae seminalis, epididymis, bladder and prostate. This showed that epithelial cells of the bladder and vesiculae seminalis also express VASA and that VASA is not germ cell specific. It was not possible to predict TESE outcome by the presence of immature germ cells. Sensitivity for this test was 47 % and specificity 59 %. Conclusion: VASA is not specifically expressed in the germ cell lineage. It is possible to detect immature germ cells in semen from men with NOA by expressing the VASA protein through immunohistochemistry. The presence of immature germ cells in semen from men with NOA cannot serve as a non-invasive parameter to predict TESE outcome.

Item Type:	Thesis (Thesis)
Supervisor name:	Driel, Dr. M.F. van
Supervisor name:	Boellaard, Drs. W.P.A. and Looijenga, Prof. Dr. L.H.J. and Erasmus Medisch Centrum and Afdeling Urologie, sectie Andrologie en Pathologie
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:41
Last Modified:	25 Jun 2020 10:41
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/267

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